Methods and compositions for genetically detecting improved milk production traits in cattle

ABSTRACT

An isolated nucleic acid molecule comprising a polymorphic site selected from the group consisting of positions 164, 269, 284, 407 and 989 of SEQ ID NO: 1, an array or a kit comprising the same. Also provided are a method for detecting single nucleotide polymorphism (SNP) in bovine proteinase inhibitor (PI) gene, a method for haplotyping a bovine cell, a method for progeny testing of cattle based on said haplotyping, a method for selectively breeding of cattle based on haplotyping a parent animal. The present invention further provides a method for testing a dairy cattle for its milk production trait, comprising haplotyping its cells, wherein a cattle having haplotypes 1, 3, 4 or 5 indicates that the cattle has desirable milk production trait. Haplotype 1 indicates that the cattle has the most desirable milk production trait.

This invention was made with United States government support awarded byUSDA/CSREES, under the grant number 05-CRHF-0-6055. The United Statesmay have certain rights in this application.

FIELD OF THE INVENTION

The present invention relates to a method of cattle progeny testingusing molecular genetic methods by assaying for the presence of at leastone genetic marker which is indicative of improved milk production andreproduction traits, including milk yield and milk composition, somaticcell score, productive life, and daughter pregnancy rate.

BACKGROUND OF THE INVENTION

Dairy cows are significant investments for dairy farmers, and enormousefforts, such as animal breeding and artificial insemination, have beenand continue to be invested in ensuring that the animals have high andsustained productivity, and that the milk produced are of high quality.A successful breeding family is the Holstein line derived from Carlin-MIvenhoe Bell. More than 25% of the highest total performance indexHolstein bulls in the United States are progenies of this individual.

Traditional breeding techniques involve the studying of sire progenies,and evaluating their milk production ratings (transmitting abilities) toguide further breeding. This standard technique requires years toevaluate the true genetic value by progeny testing each bull. Many cowsmust be bred and give birth to offspring. The females must be raised,bred, allowed to give birth and finally milked for a length of time tomeasure their phenotypic traits.

Furthermore, selection based purely on phenotypic characteristics doesnot efficiently take into account genetic variability caused by complexgene action and interactions, and the effect of the environmental anddevelopmental variants. There is thus a need for a method of geneticallyevaluating cattle to enable breeders to more accurately select animalsat both the phenotypic and the genetic level.

Marker-assisted selection can lower the high cost of progeny testingcurrently used to improve sires, since young bull progeny could beevaluated immediately after birth, and young bulls that are determinedby genetic testing to have undesirable markers would never be progenytested or even prior to birth, for the presence/absence of the marker.Therefore, there is also a need for genetic markers for improved milkproduction traits.

SUMMARY OF THE INVENTION

The present invention provides for an isolated nucleic acid moleculecomprising a polymorphic site selected from the group consisting ofpositions 164, 269, 284, 407 and 989 of SEQ ID NO: 1 and at least 17contiguous bases of SEQ ID NO: 1 adjacent to the polymorphic site,wherein the nucleic acid molecule comprises i) an adenine base atposition 164 of SEQ ID NO: 1; ii) a guanine base at position 164 of SEQID NO: 1; iii) a cytosine base at position 269 of SEQ ID NO: 1; iv) athymine base at position 269 of SEQ ID NO: 1; v) a guanine base atposition 284 of SEQ ID NO: 1; vi) a thymine base at position 284 of SEQID NO: 1; vii) a guanine base at position 407 of SEQ ID NO: 1; viii) acytosine base at position 407 of SEQ ID NO: 1; ix) a cytosine base atposition 989 of SEQ ID NO: 1; or x) a thymine base at position 989 ofSEQ ID NO: 1. It is recognized that SEQ ID NO: 1 is already known, andthe nucleic acid molecule therefore does not encompass one that consistsof SEQ ID NO: 1.

Preferably, the nucleic acid molecule which comprises at least 15, morepreferably at least 20, still more preferably at least 25, contiguousbases of SEQ ID NO: 1 adjacent to the polymorphic site. In oneembodiment, the isolated nucleic acid molecule comprises not more than1,500 nt, preferably not more than 1000 nt, more preferably not morethan 900 nt, more preferably not more than 800 nt, more preferably notmore than 700 nt, preferably not more than 600 nt, more preferably notmore than 500 nt, preferably not more than 400 nt, more preferably notmore than 300 nt, more preferably not more than 150 nt., preferably notmore than 100 nt., still more preferably not more than 50 nt.

The nucleic acid molecule preferably contains the polymorphic site whichis within 4 nucleotides of the center of the nucleic acid molecule.Preferably, the polymorphic site is at the center of the nucleic acidmolecule.

In another embodiment, the nucleic acid molecule contains thepolymorphic site which is at the 3′-end of the nucleic acid molecule.

The present invention also provides an array of nucleic acid moleculescomprising at least two nucleic acid molecules described above.

The present invention further provides a kit comprising a nucleic acidmolecule of Claim 1, and a suitable container.

Also provided is a method for detecting single nucleotide polymorphism(SNP) in bovine proteinase inhibitor (PI) gene, wherein the PI gene havea nucleic acid sequence of SEQ ID NO: 1, the method comprisingdetermining the identity of a nucleotide at position 164, 269, 284, 407or 989, and comparing the identity to the nucleotide identity at acorresponding position of SEQ ID NO: 1. Preferably, the identity of atleast two positions of positions 164, 269, 284, 407 and 989 aredetermined. More preferably, the identity of all of positions 164, 269,284, 407 and 989 are determined.

In another embodiment, the present invention provides a method forhaplotyping a bovine cell, comprising determining the identity of thenucleotides of at least two positions of 164, 269, 284, 407 and 989 ofbovine PI gene having a sequence of SEQ ID NO: 1, and comparing theidentities at the respective positions to that shown in Table 1 below.Suitable bovine cell may be an adult cell, an embryo cell, a sperm, anegg, a fertilized egg, or a zygote. The identity of the nucleotide maybe determined by sequencing the PI gene, or a relevant fragment thereof,isolated from the cell. the PI gene or a relevant fragment thereof isisolated from the cell via amplification by the polymerase chainreaction (PCR) of genomic DNA of the cell, or by RT-PCR of the mRNA ofthe cell. Preferably, the PCR or RT-PCR is conducted with a pair ofprimers selected from the group consisting of (1) SEQ ID NO: 2 and SEQID NO: 3; and (2) SEQ ID NO: 4 and SEQ ID NO: 5. In a preferredembodiment, both copies of the PI gene in the cell are haplotyped.

In a further embodiment, the present invention provides a method forprogeny testing of cattle, the method comprising collecting a nucleicacid sample from said progeny, and haplotyping said nucleic sample asdescribed above.

Further provided is a method for selectively breeding of cattle using amultiple ovulation and embryo transfer procedure (MOET), the methodcomprising superovulating a female animal, collecting eggs from saidsuperovulated female, in vitro fertilizing said eggs from a suitablemale animal, implanting said fertilized eggs into other females allowingfor an embryo to develop, and haplotyping said developing embryo, andterminating pregnancy if said developing embryo is not haplotype 1, 3, 4or 5. Preferably, pregnancy is terminated if the embryo is not haplotype1.

In a preferred embodiment, the method is used for selectively breedingdairy cattles, comprising selecting a bull that is homozygouslyhaplotype 1 and using its semen for fertilizing a female animal. Morepreferably, the female animal which is also homozygously haplotype 1.MOET procedure may be preferably used for the selective breeding.

The present invention also provides a method for testing a dairy cattlefor its milk production trait, comprising haplotyping its cells, whereina cattle having haplotype 1, 3, 4 or 5 indicates that the cattle hasdesirable milk production trait. Preferably, the test is for a cattlehaving haplotype 1 which indicates that the cattle has desirable milkproduction trait, health and reproduction traits. Haplotype 1 isassociated with high milk protein percentage, high productive life, lowsomatic cell score, and high daughter pregnancy rate. Haplotype 3 isassociated with milk fat. Haplotype 4 is associated with high milkyield, high somatic cell score and low daughter pregnancy rate.Haplotype 5 is associated with high milk yield, low fat percentage, lowprotein percentage, high somatic cell score and low daughter pregnancyrate. Thus it would be desirable to make selection decisions onhaplotype 1 that does not show any negative effects.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the PI gene sequence (SEQ ID NO: 1) where the relevantpolymorphic sites are shown.

FIG. 2 shows the novel sequence of an intron of bovine PI gene (SEQ IDNO: 6).

DETAILED DESCRIPTION OF THE INVENTION

The present inventor used the positional candidate gene approach and thepositional comparative candidate gene analysis to study the associationof the proteinase inhibitor (PI) gene with milk production andreproduction traits in Holstein dairy cattle. In principle, oncequantitative trait loci (QTL) are mapped to a chromosomal region,possible candidate genes affecting the trait of interest can beidentified. Using this approach, six haplotypes were identified (Table1), and statistically significant correlations were found to existbetween several milk production traits and the haplotypes, especiallyhaplotypes 1, 3, 4 and 5 (See Tables 4, 5 in the Examples below).Particularly, haplotype 1 has been shown to have significant correlationwith all of the following traits: milk protein percentage, productivelife of the animal, milk somatic cell score (SCS), and daughterpregnancy rate (DPR) (See Table 3 in the Examples below for details).

The present invention discloses that haplotype 1 does not show anynegative effects. Therefore it is particularly preferred to select forindividuals homozygous for haplotype 1, which would increase the effectof the haplotype. Because haplotype 1 is associated with more than onetrait at the same time, selection for haplotype 1 is equal to selectionfor multiple genetic markers. This is the first time that such a gene orgenetic marker having multiple effects is found in cattle or any otherlivestock species. TABLE 1 Haplotypes of Bovine PI Gene POSITION 164 269284 407 989 “Wild type” G C G G C Haplotype 1 (ACGCT) A C G C THaplotype 2 (GTTGT) G T T G T Haplotype 3 (GCGGT) G C G G T Haplotype 4(GTTGC) G T T G C Haplotype 5 (GCGGC) G C G G C Haplotype 6 (ACGCC) A CG C C

The term “wild-type” is used to refer to the reference coding sequencesof the PI gene as shown in FIG. 1. It has been found that specific sitesin the PI gene sequence are polymorphic. The term “polymorphism” as usedherein refers to the occurrence of two or more alternative genomicsequences or alleles between or among different genomes or individuals.“Polymorphic” refers to the condition in which two or more variants of aspecific genomic sequence can be found in a population. A “polymorphicsite” is the locus at which the variation occurs. Polymorphismsgenerally have at least two alleles, each occurring at a significantfrequency in a selected population. A polymorphic locus may be as smallas one base pair. The first identified allelic form is arbitrarilydesignated as the reference form, and other allelic forms are designatedas alternative or variant alleles. The allelic form occurring mostfrequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A biallelic polymorphism has two forms, and a triallelicpolymorphism has three forms, and so on.

Polymorphisms may provide functional differences in the geneticsequence, through changes in the encoded polypeptide, changes in mRNAstability, binding of transcriptional and translation factors to the DNAor RNA, and the like. Polymorphisms are also used to detect geneticlinkage to phenotypic variation.

One type of polymorphism, single nucleotide polymorphisms (SNPs), hasgained wide use for the detection of genetic linkage recently. SNPs aregenerally biallelic systems, that is, there are two alleles that anindividual may have for any particular SNP marker. In the instant case,the SNPs are used for determining the haplotypes of the PI gene, whichare found to have strong correlation to milk production traits.

Table 1 provides the various polymorphic sequences of the bovine PIgene. The provided sequences also encompass the complementary sequencecorresponding to any of the provided polymorphisms. In order to providean unambiguous identification of the specific site of a polymorphism,the numbering of the original PI sequence in the GenBank is shown inFIG. 1 and is used. The PI exon sequences have been published.

The present inventor sequenced an intron of the PI gene. The sequence ofthe intron is provided in FIG. 2 (SEQ ID NO: 6). This intron sequence isused to design primers PI10 which allows genomic amplification of thefragment containing the SNP at position 989.

The present invention provides nucleic acid based genetic markers foridentifying bovine animals with superior reproduction and milkproduction traits. In general, for use as markers, nucleic acidfragments, preferably DNA fragments, will be of at least 12 nucleotides(nt), preferably at least 15 nt, usually at least 20 nt, often at least50 nt. Such small DNA fragments are useful as primers for the polymerasechain reaction (PCR), and probes for hybridization screening, etc.

The term primer refers to a single-stranded oligonucleotide capable ofacting as a point of initiation of template-directed DNA synthesis underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as, DNAor RNA polymerase or reverse transcriptase) in an appropriate buffer andat a suitable temperature. The appropriate length of a primer depends onthe intended use of the primer but typically ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with a template. Theterm primer site, or priming site, refers to the area of the target DNAto which a primer hybridizes. The term primer pair means a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the DNA sequence to be amplified and a 3′, downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “probe” or “hybridization probe” denotes a defined nucleic acidsegment (or nucleotide analog segment) which can be used to identify byhybridization a specific polynucleotide sequence present in samples,said nucleic acid segment comprising a nucleotide sequence complementaryof the specific polynucleotide sequence to be identified. “Probes” or“hybridization probes” are nucleic acids capable of binding in abase-specific manner to a complementary strand of nucleic acid.

An objective of the present invention is to determine which embodimentof the polymorphisms a specific sample of DNA has. For example, it isdesirable to determine whether the nucleotide at position 164 is G or A.An oligonucleotide probe can be used for such purpose. Preferably, theoligonucleotide probe will have a detectable label, and contains an A atthe corresponding position. Experimental conditions can be chosen suchthat if the sample DNA contains an A, they hybridization signal can bedetected because the probe hybridizes to the corresponding complementaryDNA strand in the sample, while if the sample DNA contains a G, nohybridization signal is detected.

Similarly, PCR primers and conditions can be devised, whereby theoligonucleotide is used as one of the PCR primers, for analyzing nucleicacids for the presence of a specific sequence. These may be directamplification of the genomic DNA, or RT-PCR amplification of the mRNAtranscript of the PI gene. The use of the polymerase chain reaction isdescribed in Saiki et al. (1985) Science 230:1350-1354. Amplificationmay be used to determine whether a polymorphism is present, by using aprimer that is specific for the polymorphism. Alternatively, variousmethods are known in the art that utilize oligonucleotide ligation as ameans of detecting polymorphisms, for examples see Riley et al (1990)Nucleic Acids Res. 18:2887-2890; and Delahunty et al (1996) Am. J. Hum.Genet. 58:1239-1246. The detection method may also be based on directDNA sequencing, or hybridization, or a combination thereof. Where largeamounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. The nucleic acid may beamplified by PCR, to provide sufficient amounts for analysis.

Hybridization may be performed in solution, or such hybridization may beperformed when either the oligonucleotide probe or the targetpolynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Oligonucleotides may be synthesized directly on the solid supportor attached to the solid support subsequent to synthesis. Solid-supportssuitable for use in detection methods of the invention includesubstrates made of silicon, glass, plastic, paper and the like, whichmay be formed, for example, into wells (as in 96-well plates), slides,sheets, membranes, fibers, chips, dishes, and beads. The solid supportmay be treated, coated or derivatized to facilitate the immobilizationof the allele-specific oligonucleotide or target nucleic acid. Forscreening purposes, hybridization probes of the polymorphic sequencesmay be used where both forms are present, either in separate reactions,spatially separated on a solid phase matrix, or labeled such that theycan be distinguished from each other. Assays may utilize nucleic acidsthat hybridize to one or more of the described polymorphisms, and mayinclude all or a subset of the polymorphisms listed in Table 1.

Hybridization may also be performed with nucleic acid arrays andsubarrays such as described in WO 95/11995. The arrays would contain abattery of allele-specific oligonucleotides representing each of thepolymorphic sites. One or both polymorphic forms may be present in thearray, for example the polymorphism of position 164 may be representedby either, or both, of the listed nucleotides. Usually such an arraywill include at least 2 different polymorphic sequences, i.e.polymorphisms located at unique positions within the locus, and mayinclude all of the provided polymorphisms. Arrays of interest mayfurther comprise sequences, including polymorphisms, of other geneticsequences, particularly other sequences of interest. The oligonucleotidesequence on the array will usually be at least about 12 nt in length,may be the length of the provided polymorphic sequences, or may extendinto the flanking regions to generate fragments of 100 to 200 nt inlength. For examples of arrays, see Ramsay (1998) Nat. Biotech. 16:4044;Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al. (1 996)Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) NatureGenetics 14:457-460.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins whichrecognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variantalleles can be identified by single strand conformation polymorphism(SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries etal., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp.321-340, 1996) or denaturing gradient gel electrophoresis (DGGE)(Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al.,Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (WO92/15712) and the ligase/polymerase mediated geneticbit analysis (U.S. Pat. No. 5,679,524). Related methods are disclosed inWO91/02087, WO90/09455, WO95/17676, U.S. Pat. Nos. 5,302,509, and5,945,283. Extended primers containing a polymorphism may be detected bymass spectrometry as described in U.S. Pat. No. 5,605,798. Anotherprimer extension method is allele-specific PCR (Ruao et al., Nucl. AcidsRes. 17:8392, 1989; Ruao et al., Nucl. Acids Res. 19, 6877-6882, 1991;WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995). Inaddition, multiple polymorphic sites may be investigated bysimultaneously amplifying multiple regions of the nucleic acid usingsets of allele-specific primers as described in Wallace et al. (WO89/10414).

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

It is readily recognized by those ordinarily skilled in the art that inorder to maximize the signal to noise ratio, in probe hybridizationdetection procedure, the polymorphic site should at the center of theprobe fragment used, whereby a mismatch has a maximum effect ondestabilizing the hybrid molecule; and in a PCR detection procedure, thepolymorphic site should be placed at the very 3′-end of the primer,whereby a mismatch has the maximum effect on preventing a chainelongation reaction by the DNA polymerase. The location of nucleotidesin a polynucleotide with respect to the center of the polynucleotide aredescribed herein in the following manner. When a polynucleotide has anodd number of nucleotides, the nucleotide at an equal distance from the3′ and 5′ ends of the polynucleotide is considered to be “at the center”of the polynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter,” and so on.

In some embodiments, a composition contains two or more differentlylabeled oligonucleotides for simultaneously probing the identity ofnucleotides or nucleotide pairs at two or more polymorphic sites. It isalso contemplated that primer compositions may contain two or more setsof allele-specific primer pairs to allow simultaneous targeting andamplification of two or more regions containing a polymorphic site.

Alternatively, the relevant portion of the PI gene of the sample ofinterest may be amplified via PCR and directly sequenced, and thesequence be compared to the haplotype information of Table 1. In thiscase, two sets of PCR primers are preferably used for optimalamplification and to avoid the need to sequence an unnecessarily longfragment. The region that contains positions 164-407 can be amplifiedwith a first set of primers (e.g. SEQ ID NO: 2 and 3), and the regioncontaining position 989 can be amplified separately with a second set ofprimers (e.g. SEQ ID NO: 4 and 5). It is readily recognized thatnumerous other primers can be devised to achieve the same objectives.The sequence information will allow the determination of all sixpolymorphic site shown in Table 1. PCR and sequencing techniques arewell known in the art and reagents and equipments are readily availablecommercially.

DNA markers have several advantages; segregation is easy to measure andis unambiguous, and DNA markers are co-dominant, i.e., heterozygous andhomozygous animals can be distinctively identified. Once a marker systemis established selection decisions could be made very easily, since DNAmarkers can be assayed any time after a blood sample can be collectedfrom the individual infant animal, or even earlier by testing embryos invitro if very early embryos are collected. The use of marker assistedgenetic selection will greatly facilitate and speed up cattle breedingproblems. For example, a modification of the multiple ovulation andembryo transfer (MOET) procedure can be used with genetic markertechnology. Specifically, females are superovulated, eggs are collected,in vitro fertilized using semen from superior males and implanted intoother females allowing for use of the superior genetics of the female(as well as the male) without having to wait for her to give birth toone calf at a time. Developing blastomeres at the 4-8 cell stage may beassayed for presence of the marker, and selection decisions madeaccordingly.

In one embodiment of the invention an assay is provided for detection ofpresence of a desirable genotype and/or haplotype using the markers.

The term “genotype” as used herein refers to the identity of the allelespresent in an individual or a sample. In the context of the presentinvention a genotype preferably refers to the description of thepolymorphic alleles present in an individual or a sample. The term“genotyping” a sample or an individual for a polymorphic marker refersto determining the specific allele or the specific nucleotide carried byan individual at a polymorphic marker.

The term “haplotype” refers to the actual combination of alleles on onechromosome. At the DNA level, it refers to a sequence of nucleotidesfound at two or more polymorphic sites in a locus on a singlechromosome. As used herein, haplotype includes a full-haplotype and/or asub-haplotype. Full-haplotype is the 5′ to 3′ sequence of nucleotidesfound at all polymorphic sites examined in a locus on a singlechromosome from a single individual, while sub-haplotype refers to the5′ to 3′ sequence of nucleotides seen at a subset of the polymorphicsites examined in a locus on a single chromosome from a singleindividual. Relatedly, the term “haplotype pair” refers to the twohaplotypes found for a locus in a single individual. “Haplotyping” is aterm for a process for determining one or more haplotypes in anindividual and includes use of family pedigrees, molecular techniquesand/or statistical inference.

The present invention is suitable for identifying a bovine, including ayoung or adult bovine animal, an embryo, a semen sample, an egg, afertilized egg, or a zygote, or other cell or tissue sample therefrom,to determine whether said bovine posseses one of the haplotypes of thepresent invention, some of which are indicative of improved milkproduction traits.

The method preferably is for haplotyping the bovine PI gene, whichcomprises identifying the sequence of nucleotides at positions 164, 269,284, 407, and 989, for at least one copy of the PI gene and assigning tothe individual a bovine PI haplotype, wherein the bovine PI haplotype isselected from the group consisting of the haplotypes shown in Table 1.The method may be used to identify the haplotype of both copies the PIgene in the animal, and assigning a haplotype pair to the animal.

One embodiment of a haplotyping method of the invention comprisesexamining one copy of the PI gene, or a fragment thereof, to identifythe nucleotide at two or more polymorphic sites in that copy to assign ahaplotype to the individual.

As will be readily appreciated by those skilled in the art, if a PI geneis cloned and sequenced any individual clone will typically only providehaplotype information on one of the two PI gene copies present in anindividual. If haplotype information is desired for the individual'sother copy, additional PI clones will usually need to be examined.Typically, at least five clones should be examined to have more than a90% probability of haplotyping both copies of the PI gene in anindividual.

Further provided is a method for genotyping the bovine PI gene,comprising determining for the two copies of the PI gene present theidentity of the nucleotide pair at one or more polymorphic sites atpositions 164, 269, 284, 407, and 989, wherein the one or morepolymorphic sites (PS) have the position and alternative alleles shownin Table 1.

One embodiment of a genotyping method of the invention involvesexamining both copies of the PI gene, or a fragment thereof, to identifythe nucleotide pair at one or more polymorphic sites listed in Table 1in the two copies to assign a genotype to the individual. In someembodiments, “examining a gene” may include examining one or more of:DNA containing the gene, mRNA transcripts thereof, or cDNA copiesthereof. As will be readily understood by the skilled artisan, the two“copies” of a gene, mRNA or cDNA, or fragment thereof in an individualmay be the same allele or may be different alleles. In anotherembodiment, a genotyping method of the invention comprises determiningthe identity of the nucleotide pair at each of the polymorphic sitelisted in Table 1.

The present invention further provides a kit for haplotyping a bovinesample, the kit comprising in a container a nucleic acid molecule, asdescribed above, designed for detecting the one or more of thepolymorphisms listed in Table 1, and optionally at least anothercomponent for carrying out such detection. Preferably, a kit comprisesat least two oligonucleotides packaged in the same or separatecontainers. The kit may also contain other components such ashybridization buffer (where the oligonucleotides are to be used as aprobe) packaged in a separate container. Alternatively, where theoligonucleotides are to be used to amplify a target region, the kit maycontain, preferably packaged in separate containers, a polymerase and areaction buffer optimized for primer extension mediated by thepolymerase, such as PCR.

In one embodiment the present invention provides a breeding methodwhereby haplotyping as described above is conducted on bovine embryos,and based on the results, certain cattle are either selected or droppedout of the breeding program. Most preferably, individuals carryinghaplotype 1 is selected. The unexpected results of the present inventionshow that animals carrying haplotype 1 has improved milk productiontraits, including low SCS, high DPR and high productivity, as shown inTable 3. Haplotype 3 is positively correlated with fat yield. Haplotype4 is positively correlated with milk yield and SCS and negativelycorrelated with DPR, and haplotype 5 is positively correlated with milkyield and SCS, and negatively correlated with fat percentage and proteinpercentage (tables 4, 5).

Through use of the linked marker loci, the different haplotypes can bemanipulated in genetic improvement programs by procedures termed “markerassisted selection” (MAS), for genetic improvement within a breedingnucleus; or “marker assisted introgression” for transferring usefulalleles from a resource population to a breeding nucleus (Soller 1990;Soller 1994).

The following examples are intended to illustrate preferred embodimentsof the invention and should not be interpreted to limit the scope of theinvention as defined in the claims.

EXAMPLES Example 1 Identification of Haplotypes and Determination ofTheir Association with Milk Production Traits

Resource Population and Phenotypic Data

The Cooperative Dairy DNA Repository (CDDR) is an extension of the DairyBull DNA Repository (DBDR) started at the University of Illinois in1993. The DBDR was established to identify QTL in large Holsteinfamilies using the granddaughter design (Weller et al. 1990). In thegranddaughter design, QTL are mapped using genotypes from grandsires andtheir sons and the granddaughters' phenotypic values as the traitendpoints. QTL can be mapped and markers flanking the QTL can beidentified. Once flanking markers have been identified, marker-assistedselection can be utilized to shorten the generation interval and reducetime and cost of progeny testing.

Thirty seven (37) half-sib families comprised of 2,363 sons wereselected from the CDDR collection for quantitative trait gene (QTG)detection using the granddaughter design. Data for PredictedTransmitting Abilities (PTA) of the traits of interest (protein yield,protein percentage, fat yield, fat percentage, milk yield, somatic cellscore, daughter pregnancy rate, productive life) were obtained from theAnimal Improvement Programs Laboratory (AIPL-USDA). The PTA includes thedeviation of the daughter performance from the population mean adjustedfor the genetic merit of mate and the genetic merit of the grandsire andgrand dam (Van-Raden and Wiggans, 1991).

Previous efforts of QTL mapping on cattle chromosome 21 have revealeddifferent QTL affecting production and health traits. Hayen et al.(1999) reported a putative QTL affecting milk yield and protein yield inlinkage with the macrosatellite marker D21S27 at position 56 ofchromosome 21. Rodriguez-Zas et al. (2002) reported that a QTL locatedat position 56 was associated with variations on somatic cell score(SCS) and protein yield. Mosig et al. (2001) reported a QTL thataffected protein percentage at position 67.3. Accordingly, candidategenes in the chromosomal region of 10 cM, from position 57-67 werechosen for further investigation.

This region contains four characterized genes: CHGA, PI, AACT, andSERPINA3. The PI gene is further specifically chosen for investigationof its effect on milk production traits, because it has been reportedthat the PI protein is present in human milk and it might increase thesurvival of milk proteins by various mechanisms (Chowanadisai andLonnerdal, 2002).

Polymorphism in PI Gene

It is necessary to determine whether polymorphism exists in this gene.Toward that goal, we amplified the total cDNA sequence of the gene froma wide range of cattle tissues. The complete cDNA sequence of bovinealpha 1-antitrypsin, or the proteinase inhibitor (PI) gene is known, andis available in the gene bank (Accession # X63129, Sinha et al., 1992,Biochim. Biophys. Acta 1130 (2), 209-212). The sequence (SEQ ID NO: 5)is shown in FIG. 1. Four primers based on the known PI sequence weredesigned. These primers are: P17 5′-ATGGCACTCTCCATCACGCG 3′ (SEQ ID NO:2) PI11 5′-CCACTAGCTTTGCACTCTCA 3′ (SEQ ID NO: 3) PI95′-TTGGACACCTTCAGAGGCTG 3′ (SEQ ID NO: 4) PI10 5′-AGTGTGAGAGCACGGGGAGA3′ (SEQ ID NO: 5)

We amplified the total cDNA sequence of the gene from a wide range ofcattle tissues. RT-PCR products from a wide range of tissues from fivefetuses and five cows were analyzed by direct sequencing. The tissueswere: heart, brain, lung, muscle, liver, kidney, pancreas, bone,cartilage, spleen, adrenal, mammary gland and ovary. Five polymorphicSNPs at positions 164, 269, 284,407, and 989 were identified. Sixdifferent haplotypes could be determined from the five SNPs (See FIG. 1and Table 1).

Selective Genotyping

Semen samples of 37 half-sib families comprised of 2,363 sons wereselected from the CDDR collection for quantitative trait gene (QTG)detection using the granddaughter design.

For protein percentage trait, we used the selective DNA genotypingapproach in order to reduce the costs of screening the population forpolymorphic markers. In this approach, determination of associationbetween a genetic marker and QTG is based on the distribution of themarker alleles among the samples of the extreme high and low phenotypicgroups. Within each sire family, we choose 10% of the sons with highestPTAs for protein percentage and 10% of the sons with the lowest PTAs. Atotal of 423 individuals were chosen for selective genotyping analysisfor protein percentage trait.

To search for associations with other traits of interest (see Table 3),we genotyped 1,258 individuals.

Single Nucleotide Polymorphism (SNP) Detection

Genomic DNA was extracted from semen samples with phenol/chloroform andproteinase k procedures (Kappes et al. 2000). The DNA concentration wasmeasured using spectrophotometer Pro 2.1 (Pharmacia).

Primers were designed in the PI gene to amplify the total cDNA sequenceof the gene. In order to detect polymorphisms in the PI gene exons, weextracted total RNA from a wide range of tissues by using RT-PCR.

RNA Extraction.

Cattle tissues were obtained from a local slaughterhouse. Afterdissection, tissues were immediately chilled on ice and submerged in anappropriate volume of RNALater RNA stabilization reagent (QIAGEN). TotalRNA was extracted using the RNeasy kit (QIAGEN). The protocol for totalRNA isolation from heart and muscle tissues was modified from thestandard protocol for other tissues, due to the abundance of contractileproteins and collagen.

Sequencing of PCR and RT-PCR Products.

The sizes of PCR and RT-PCR products were estimated on a 1% agarose gel.The products were purified from the PCR solution, using GFX PCR DNAPurification Kit (Amersham Biosciences). Sequencing reactions consistedof 2 ul of BigDye Terminator mix (Applied Biosystems), 6 ul of dilutionbuffer (200 mM Tris HCl pH 9.0, 5 mM MgC12), 5 pmol of primer, and 0.1ug of template DNA in a final reaction volume of 20 ul. Cycle conditionswere an initial denaturation at 96° for 3′, then 50 cycles of 96° for10″, 58° for 4′, followed by 7′ at 72°. Excess dye terminators wereremoved using CleanSeq magnetic bead sequencing reaction clean up kitfrom Agencourt Biosciences. The samples were resuspended off of thebeads in 50 ul of ddH2O. 10 ul of each sample was loaded into a 96 wellPCR plate and loaded onto the sequencers according to the manufacturer'sinstructions. Samples were electrophoresed on an Applied Biosystems3730XL automated DNA sequencing instrument, using 50 cm capillary arraysand POP-6 polymer. Data were analyzed using Applied Biosystems version5.0 of Sequencing Analysis. SNPs were identified by visually inspectingeach base in sequencing traces.

Inferring Haplotypes

Haplotypes were inferred as follows (Lagziel et al. 1996):

-   -   1. From homozygous individuals. For example, from an individual        showing genotypes G/G, T/T, T/T, G/G, C/C, the haplotype GTTGC        (HAPLOTYPE 4) was inferred;    -   2. From heterozygous individuals showing only a single        heterozygous site. For example, from an individual having the        genotype A/A, C/C, G/G, G/G, C/T, haplotypes ACGGC and ACGGT        were inferred;    -   3. From direct sequencing of 30 sires. As shown in Table 2, five        sires were homozygous for the five SNPs and 25 sires were        heterozygous for at least one SNP. Six different haplotypes        could be determined from the five SNPs; and    -   4. From direct sequencing of heterozygote and homozygote sons        within each family. A total of 100 sons were sequenced.        Statistical Analysis

Analysis of variance (ANOVA) for each haplotype and trait combinationwas performed using the PROC GLM function of SAS (SAS Institute, Cary,N.C.). TABLE 2 Genotypes and Haplotypes of 30 Sires as Determined byDirect Sequencing SIRE 164 269 284 407 989 HAPLOTYPE I HAPLOTYPE II 1A/G C G G/C C/T ACGCT (HAPLOTYPE 1) GCGGC (HAPLOTYPE 5) 2 G I T G C/TGTTGT (HAPLOTYPE 2) GTTGC (HAPLOTYPE 4) 3 G T T G C/T GTTGT (HAPLOTYPE2) GTTGC (HAPLOTYPE 4) 4 A/G C G G/G C/T ACGCT (HAPLOTYPE 1) GCGGC(HAPLOTYPE 5) 5 A/G C G G/C C/T ACGCT (HAPLOTYPE 1) GCGGC (HAPLOTYPE 5)6 A/G C/T G/T G/G C/T ACGCT (HAPLOTYPE 1) GTTGC (HAPLOTYPE 4) 7 A/G C/TG/T G/C C/T ACGCT (HAPLOTYPE 1) GTTGC (HAPLOTYPE 4) 8 A/G C G G/G C/TACGCT (HAPLOTYPE 1) GCGGC (HAPLOTYPE 5) 9 A C G C C/T ACGCT(HAPLOTYPE 1) ACGCC (HAPLOTYPE 6) 10 G C/T G/T G C/T GCGGT (HAPLOTYPE 3)GTTGC (HAPLOTYPE 4) 11 A C G C C/T ACGCT (HAPLOTYPE 1) ACGCC (HAPLOTYPE6) 12 G C/T G/T G C/T GTTGT (HAPLOTYPE 2) GCGGC (HAPLOTYPE 5) 13 G C G GC/T GCGGT (HAPLOTYPE 3) GCGGC (HAPLOTYPE 5) 14 A/G C/T G/T G/G C/T ACGCT(HAPLOTYPE 1) GTTGC (HAPLOTYPE 4) 15 A/G C/T G/T G/C C/T ACGCT(HAPLOTYPE 1) GTTGC (HAPLOTYPE 4) 16 A C G C C/T ACGCT (HAPLOTYPE 1)ACGCC (HAPLOTYPE 6) 17 A/G C/T G/T G/C C/T ACGCT (HAPLOTYPE 1) GTTGC(HAPLOTYPE 4) 18 A/G C G G/C C/T ACGCT (HAPLOTYPE 1) GCGGC (HAPLOTYPE 5)19 G T T G C/T GTTGT (HAPLOTYPE 2) GTTGC (HAPLOTYPE 4) 20 A/G C G G/CC/T ACGCT (HAPLOTYPE 1) GCGGC (HAPLOTYPE 5) 21 G C G G C/T GCGGT(HAPLOTYPE 3) GCGGC (HAPLOTYPE 5) 22 G/A C/T G/T G/C C/T ACGCT(HAPLOTYPE 1) GTTGC (HAPLOTYPE 4) 23 G C G G C GCGGC (HAPLOTYPE 5) GCGGC(HAPLOTYPE 5) 24 A C G C C/T ACGCT (HAPLOTYPE 1) ACGCC (HAPLOTYPE 6) 25G C/T G/T G C GTTGC (HAPLOTYPE 4) GCGGC (HAPLOTYPE 5) 26 G T T G C GTTGC(HAPLOTYPE 4) GTTGC (HAPLOTYPE 4) 27 G C/T G/T G C GTTGC (HAPLOTYPE 4)GCGGC (HAPLOTYPE 5) 28 G T T G C GTTGC (HAPLOTYPE 4) GTTGC (HAPLOTYPE 4)29 G T T G C GTTGC (HAPLOTYPE 4) GTTGC (HAPLOTYPE 4) 30 A C G C T ACGCT(HAPLOTYPE 1) ACGCT (HAPLOTYPE 1)

Analysis was performed for the combined data from all familiessegregating with same haplotype.

The average allele substitution effects (α) were calculated followingthe method of Falconer and MacKay (1996) using:α=a+d(q−p)where a and d are the homozygous and heterozygous genotypic values,respectively, and q and p are the allele frequencies of either of thetwo alleles of a bi-allelic polymorphic site of the gene.

The various traits are defined and measured according to the USDAstandards set by the Animal Improvement Programs Laboratory (AIPL) ofthe United States Department of Agriculture. The total milk yield ismeasured in pounds (lb). Milk fat and protein content are measured aspercentages. Productive life (PL) means duration of a cow in the milkingherd before removal by voluntary, involuntary culling, or death.PL=Total months in milk limited to 10 months per lactation and 84 monthsof age. Somatic Cell Score=log₂ (SCC, 100,000)+3; where SCC is somaticcells per milliliter. SCS of 3 is equal to 100,00 cells/ml. Lowest SCSis associated with lowest rates of mastitis infection (Schutz, 1994).Daughter Pregnancy Rate (DPR)=the percentage of non-pregnant cows thatbecome pregnant during each 21-day period. A DPR of 1.0 implies thatdaughters are 1% more likely to become pregnant during a given 21 dayestrus cycle than daughters of a bull with an evaluation of zero. Anincrease of 1% in PTA DPR equals a decrease of 4 days in PTA days open.

Results are shown in Tables 3 and 4. In Table 2 we presented genotypingand haplotyping results of the 30 available sires. As shown in Table 2,sires 1, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 20, 22, 24, and 30share haplotype 1 (ACGCT). A total of 759 sons of those sires wereincluded in the analysis of haplotype 1. Table 3 shows the ANOVAanalysis of these 17 sire families. Since all sons share one commonhaplotype (ACGCT), we determined haplotypes in these sons according to asingle genotype at position 989. Three possible genotypes weredesignated: TT for individuals homozygous at position 989 (and byinference are also homozygous for haplotype 1), CT for individualsheterozygous at position 989 (also heterozygous for haplotype 1), CC forindividuals homozygous for other haplotypes. Table 3 also shows the meangenetic values of the different genotypes TT, CT, and CC. Table 4 showsthe ANOVA analysis of all haplotypes with significant effects. Table 5shows the allele substitution effects found associated with thedifferent haplotypes (Falconer, 1996). The signs + and − indicatewhether the effect of the haplotype is positive or negative. It isnoteworthy that negative effects on SCS are desirable, since lowest SCSis associated with lowest rates of mastitis infection.

Example 2 Experimental Design for Identification of Haplotypes

The following is exemplifies experimental designs for determininghaplotypes of a sample. Genomic DNA from the sample may be firstamplified via PCR with primers PI9, PI10 followed with restrictionenzyme RsaI. This enzyme digests C allele only at position 989, so thatTT products would not be digested, while CT and CC products would bedigested.

Haplotype 1 can then be differentiated from 2 and 3 by specific primeramplification at position 164, by designing a primer that ends with A orG.

Haplotype 2 can be differentiated from 3 by specific primeramplification at position 269 or 285.

Haplotype 6 can be differentiated from 4 and 5 by position 164.

Haplotype 4 can be differentiated from 5 by positions 269 and 285. TABLE3 Comparison of Various Traits Between Haplotype 1 (TT) and OtherHaplotypes ANOVA Contrast Regression analysis Mean/genotype analysisanalysis Trait P TT CT CC TT vs. others CC vs. others p PTA milk 0.8119443.18 480.15 450.89 0.73 0.87 0.8914 PTA fat 0.6194 19.084 17.13517.929 0.44 0.93 0.6094 PTA fat % 0.2494 0.01455 0.0005 0.008 0.24 0.950.4882 PTA prot. 0.21818 21.689 19.492 18.255 0.1153 0.2110 0.1194 PTAprot. % 0.0013 0.036 0.023 0.020 0.0003 0.042 0.0015 PTA PL¹ <0.00010.4078 0.2160 −0.2248 <0.0001 <0.0001 <0.0001 PTA SCS² <0.0001 3.09233.1073 3.1836 0.0003 <0.0001 <0.0001 PTA DPR³ 0.0103 0.315 0.176 −0.0090.0069 0.0045 0.0027 DYD milk 0.5423 397.23 461.01 397.9 0.65 0.670.9274 DYD fat 0.747 18.294 16.579 16.731 0.476 0.772 0.5625 DYD fat %0.181 0.0185 0.0015 0.0117 0.222 0.866 0.4948 DYD milk prot. 0.478388.69 451.85 375.53 0.723 0.549 0.9580 DYD prot. 0.2174 20.27 18.4416.05 0.11 0.10 0.0822 DYD prot. % 0.0014 0.0371 0.0215 0.0215 0.00050.101 0.0035 DD⁵ SCS <0.0001 −0.0066 0.0127 0.107 0.0016 <0.0001 <0.0001DD DPR 0.1961 0.0396 0.0169 0.0076 0.071 0.210 0.0847¹Productive life;²somatic cell score;³daughter pregnancy rate;⁴daughter yield deviation;⁵daughter deviation

TABLE 4 ANOVA Analysis (p Values) of All haplotypes Affecting DifferentTraits Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4 Haplotype 5Haplotype 6 Trait (ACGCT) (GTTGT) (GCGGT) (GTTGC) (GCGGC) (ACGCC) (PTA)N = 759 N = 184 N = 130 N = 455 N = 447 N = 123 Milk Yield 0.0232 0.0810.0035 Fat Yield 0.0182 Milk Fat % 0.0095 Protein Yield 0.0234 0.0920Milk Protein % 0.0013 0.0069 Productive Life <0.0001 0.0240 SCS <0.00010.0323 0.0241 0.0303 DPR 0.0103 0.0051 DYD milk 0.0116 0.0291 0.0030 DYDfat DYD fat % 0.0296 DYD milk prot. 0.0106 0.0290 0.0054 DYD protein0.0199 DYD protein % 0.0014 0.0198 DD productive life <0.0001 0.04780.0387 DD SCS <0.0001 0.0764 0.0335 DD DPR 0.0276

TABLE 5 Allele Substitution Effects of All Haplotypes And Selected MilkProduction Traits Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4Haplotype 5 Haplotype 6 (ACGCT) (GTTGT) (GCGGT) (GTTGC) (GCGGC) (ACGCC)Trait N = 759 N = 184* N = 130* N = 455 N = 447 N = 123* Milk Yield (−)199.00 (+)    380 (+) Fat Yield (+) Milk Fat %  0.044 (+) −0.0294 (−)Protein Yield (−) Milk Protein %  0.012 (+)  0.013 (−)  −0.019 (−)Productive Life 0.6974 (+)  0.318 (−) (−) SCS 0.1074 (−) 0.0492 (+) (+)DPR 0.3366 (+)  −0.460 (−) (−) DD productive life  −0.360 (−)*Due to small number of individuals, allele substitution effects valueswere not included.(+), positive effect;(−), negative effect.References:

-   1. Weller, J., Kashi, Y. and Soller, M. (1990). Daughter and    granddaughter design for mapping of quantitative trait loci in dairy    cattle. J. Dairy Sci. 73:2525-2537.-   2. VanRaden, P. M., and Wiggans, G. R. (1991). Derivation,    calculation, and the use of National Model Information. J. Dairy Sc.    74:2737-2746-   3. Kappes, S. M., Bennett, G. L., Keele, J. W., Echternkamp, S. E.,    Gregory, K. E. and Thallman. R. M. (2000). Initial results of    genomic scans for ovulation rate in a cattle population selected for    increased twinning rate. J Anim Sci. 78:3053-3059.-   4. Lagziel, A., Lipkin, E. and Soller, M. (1996). Association    between SCCP haplotypes at the bovine growth hormone gene and milk    protein percentage. Genetics 142:945-951.-   5. Falconer, D. S. and Mackay F. C. (1996). Introduction to    Quantitative Genetics. 4th ed. Longman Scientific and Technical, New    York.-   6. Heyen, D. W., Weller, J. I., Ron, M., Band, M. and Beever J. E.    et al. (1999). A genome scan for QTL influencing milk production and    health traits in dairy cattle. Physiol. Genomics 1:165-175.-   7. Rodriguez-Zas, S. L., Southey, B. R., Heyen, D. W. and Lewin H A    (2002). Interval and composite interval mapping of somatic cell    score, yield, and components of milk in dairy cattle.J Dairy Sci.    85:3081-3091.-   8. Mosig, M. O., Lipkin, E., Khutoreskaya, G., Tchourzyna, E.,    Soller, M. and Friedmann A. (2001). A whole genome scan for    quantitative trait loci affecting milk protein percentage in    Israeli-Holstein cattle, by means of selective milk DNA pooling in a    daughter design, using an adjusted false discovery rate criterion.    Genetics. 157:1683-98.-   9. Chowanadisai, W. and Lonnerdal, B. (2002). Alpha(1)-antitrypsin    and antichymotrypsin in human milk: origin, concentrations, and    stability. Am J Clin Nutr. 76:828-833.-   10. Soller, M. (1990) Genetic mapping of the bovine genome using    DNA-level markers with particular attention to loci affecting    quantitative traits of economic importance. J. Dairy Sci.    73:2628-2646.-   11. Soller, M. (1994) Marker-assisted selection, an overview. Anim.    Biotech. 5:193-208.-   12. Schutz, M. (1994) Genetic evaluation of somatic cell scores for    United States dairy cattle. J. D. Sci. 77:2113-2129

1. An isolated nucleic acid molecule comprising a polymorphic siteselected from the group consisting of positions 164, 269, 284, 407 and989 of SEQ ID NO: 1 and at least 17 contiguous bases of SEQ ID NO: 1adjacent to the polymorphic site, wherein the nucleic acid moleculecomprises i) an adenine base at position 164 of SEQ ID NO: 1; ii) aguanine base at position 164 of SEQ ID NO: 1; iii) a cytosine base atposition 269 of SEQ ID NO: 1; iv) a thymine base at position 269 of SEQID NO: 1; v) a guanine base at position 284 of SEQ ID NO: 1; vi) athymine base at position 284 of SEQ ID NO: 1; vii) a guanine base atposition 407 of SEQ ID NO: 1; viii) a cytosine base at position 407 ofSEQ ID NO: 1; ix) a cytosine base at position 989 of SEQ ID NO: 1; or x)a thymine base at position 989 of SEQ ID NO: 1; or a nucleic acidmolecule that is fully complementary to a nucleic acid sequence of(i)-(x), provided that the a nucleic acid molecule is not one consistingof SEQ ID NO:
 1. 2. A nucleic acid molecule according to claim 1, whichcomprises at least 15 contiguous bases of SEQ ID NO: 1 adjacent to thepolymorphic site.
 3. A nucleic acid molecule according to claim 1, whichcomprises at least 20 contiguous bases of SEQ ID NO: 1 adjacent to thepolymorphic site.
 4. An isolated nucleic acid molecule according toclaim 1, which comprises not more than 150 nt.
 5. An isolated nucleicacid molecule according to claim 1, which comprises not more than 100nt.
 6. An isolated nucleic acid molecule according to claim 1, whichcomprises not more than 50 nt.
 7. A nucleic acid molecule according toclaim 1, wherein the polymorphic site is within 4 nucleotides of thecenter of the nucleic acid molecule.
 8. A nucleic acid moleculeaccording to claim 7, wherein the polymorphic site is at the center ofthe nucleic acid molecule.
 9. A nucleic acid molecule according to claim1, wherein the polymorphic site is at the 3′-end of the nucleic acidmolecule.
 10. An array of nucleic acid molecules comprising at least twonucleic acid molecules according to claim
 8. 11. A kit comprising anucleic acid molecule of claim 1, and a suitable container.
 12. A methodfor detecting single nucleotide polymorphism (SNP) in bovine proteinaseinhibitor (PI) gene, wherein the PI gene have a nucleic acid sequence ofSEQ ID NO: 1, the method comprising determining the identity of anucleotide at position 164, 269, 284, 407 or 989, and comparing theidentity to the nucleotide identity at a corresponding position of SEQID NO:
 1. 13. A method according to claim 12, wherein the identity of atleast two positions of positions 164, 269, 284, 407 and 989 aredetermined.
 14. A method according to claim 12, wherein the identity ofall of positions 164, 269, 284, 407 and 989 are determined.
 15. A methodfor haplotyping a bovine cell, comprising determining the identity ofthe nucleotides of at least two positions of 164, 269, 284, 407 and 989of bovine PI gene having a sequence of SEQ ID NO: 1, and comparing theidentities at the respective positions to that shown in the table below:POSITION 164 269 284 407 989 Wild type G C G G C Haplotype 1 (ACGCT) A CG C T Haplotype 2 (GTTGT) G T T G T Haplotype 3 (GCGGT) G C G G THaplotype 4 (GTTGC) G T T G C Haplotype 5 (GCGGC) G C G G C Haplotype 6(ACGCC) A C G C C

thereby determining the haplotype.
 16. A method according to claim 15,wherein the bovine cell is an adult cell, an embryo cell, a sperm, anegg, a fertilized egg, or a zygote.
 17. A method according to claim 15,wherein the identity of the nucleotide is determined by sequencing thePI gene, or a relevant fragment thereof, isolated from the cell.
 18. Amethod according to claim 17, wherein the PI gene or a relevant fragmentthereof is isolated from the cell via amplification by the polymerasechain reaction (PCR) of genomic DNA of the cell, or by RT-PCR of themRNA of the cell.
 19. A method according to claim 17, wherein the PCR orRT-PCR is conducted with a pair of primers selected from the groupconsisting of (1) SEQ ID NO: 2 and SEQ ID NO: 3; and (2) SEQ ID NO: 4and SEQ ID NO:
 5. 20. A method according to claim 17, wherein bothcopies of the PI gene in the cell are haplotyped.
 21. A method forprogeny testing of cattle, the method comprising collecting a nucleicacid sample from said progeny, and haplotyping said nucleic sampleaccording to claim
 15. 22. A method for selectively breeding of cattleusing a multiple ovulation and embryo transfer procedure (MOET), themethod comprising superovulating a female animal, collecting eggs fromsaid superovulated female, in vitro fertilizing said eggs from asuitable male animal, implanting said fertilized eggs into other femalesallowing for an embryo to develop, and haplotyping said developingembryo according to claim 15, and terminating pregnancy if saiddeveloping embryo is not haplotype 1, 3, 4 or
 5. 23. A method accordingto claim 22, wherein pregnancy is terminated is said embryo is nothaplotype
 1. 24. A method for selectively breeding dairy cattles,comprising selecting a bull that is homozygously haplotype 1 and usingits semen for fertilizing a female animal.
 25. A method according toclaim 24, wherein the female animal is in vitro fertilized.
 26. A methodaccording to claim 24, wherein MOET procedure is used.
 27. A methodaccording to claim 24, wherein said female animal is also homozygouslyhaplotype
 1. 28. A method for testing a dairy cattle for its milkproduction trait, comprising haplotyping its cells according to claim15, wherein a cattle having haplotype 1,3, 4 or 5 indicates that thecattle has desirable milk production trait.
 29. A method according toclaim 28, wherein a cattle having haplotype 1 indicates that the cattlehas a desirable milk production, health or reproduction trait.